1. Field of the Invention
The present invention concerns an assay for quantitating human DNA, particularly quantitating human DNA present in samples containing extraneous nonhuman DNA.
2. Description of the Related Art
The quantitative detection of bio-materials in mixed forensic samples has been approached using a variety of different systems. Early approaches to identify the origin of mixed sample components involved the use of high-performance liquid chromatography (HPLC) based methods. See H. F. Inoue et al., Species Identification of Blood and Bloodstains by High-Performance Liquid Chromatography, INT. J. LEGAL MED. 104:9-12 (1990). These methods have proven useful, although the detection limits using these approaches are restrictive. The detection of single copy nuclear DNA (Deoxyribonucleic acid) sequences has also been useful in this regard, but is limited as a result of their single copy. Polymerase chain reaction (PCR) based analysis of mitochondrial DNA sequences has also been used in the analysis of complex DNA samples. The advantage of mitochondrial based DNA analyses derives from the fact that there are many mitochondria per cell, and many mitochondrial DNA molecules within each mitochondria making mitochondrial DNA a naturally amplified source of genetic variation. See R. L. Cann et al., Mitochondrial DNA and Human Evolution, NATURE 325:31-36 (1987). However, a significant proportion of human forensic casework involves the analysis of nuclear loci. See R. Chakrabarty et al., The Utility of Short Tandem Repeat Loci beyond Human Identification: Implications for Development of New DNA Typing Systems, ELECTROPHORESIS 20:1682-1696 (1999), making the identification and quantitation of human nuclear DNA a paramount issue.
Commercially available products for human DNA quantitation include the Quantiblot® (Applied Biosystems, Inc.) and the AluQuant® (Promega Corporation) systems. The Quantiblot® system is based on the hybridization of a biotinylated oligonucleotide probe to extracted DNA, followed by visual comparison of the colorimetric or chemiluminescent sample results to the DNA standards. The AluQuant® system utilizes a luciferase reaction that results in light output suitable for interpretation with a luminometer. M. N. Mandrekar et al., Development of a Human DNA Quantitation System, CROAT. MED. J. 42: 336-339 (2001).
These systems can become quite costly, particularly if a luminometer needs to be purchased. The Quantiblot® and AluQuant® systems also detect non-human primate DNA, as well as human DNA, and the detection limit for each of these systems is approximately 0.1 ng (nanogram). These proprietary systems use technology that is not known to the public, because it is apparently being maintained as a trade secret. Although Promega uses the trademark “AluQuant®,” it is not known whether the technology actually uses Alu elements, and there is considerable doubt in this regard because the system detects non-human DNA, which is not consistent with the best Alu-based technology.
The use of Alu PCR amplification has been proposed as a more sensitive method for the quantitation of genomic DNA compared to typical blot-based procedures currently used in most forensic laboratories. See M. E. Sifis et al., A More Sensitive Methodfor the Quantitation of Genomic DNA by Alu Amplification, J. FORENSIC SCI. 47:589-592 (2002). Note that the term “AluQuant” is a trademark of Promega Corporation and the AluQuant® system may not necessarily be based on Alu mobile elements, per se.
Alu elements are transposable DNA elements which have amplified to a copy number of over 1 million elements throughout primate evolution, thus producing a series of subfamilies of Alu elements that appear to be of different genetic ages. The expansion of these elements throughout primate evolution has created several recently integrated “young” Alu subfamilies that are present in the human genome but are largely absent from non-human primates. M. A. Batzer and P. L. Deininger, Alu Repeats and Human Genomic Diversity, NAT. REV. GENET. 3:370-379 (2002). These human-specific subfamilies only have a fraction of the copy number compared to primate-specific elements, however, so that they are relatively less available for assay use.
Recently Sifits, et al., supra, reported a method whereby a fluorescently labeled oligonucleotide PCR primer pair was designed to amplify a generic Alu sequence within primate DNA. They reported that the assay had a sensitivity level of 100-2.5 pg of DNA, which is an improvement over other assays with detection limits of 100-150 pg. However, their assay did not distinguish human from non-human DNA in forensic samples that were contaminated with non-human DNA, which frequently occurs.
Therefore, a need exists for a sensitive assay capable of quantitating human DNA present in samples also containing extraneous non-human DNA.